Biofilms are matrix-enclosed microbial assemblies adhering to non-biological and biological surfaces. They undergo dynamic environment-dependent changes. Many biofilms constitute complex microbial communities rather than assemblies composed of one or a few species. Species uncultivable under most in vitro growth conditions may contribute to these biofilms. The most frequently occurring biofilm-associated infection world- wide is the urinary tract infection (UTI). Nosocomial indwelling catheter-associated urinary tract infections (CA- UTI) are contracted by more than 1 million patients per year in the U.S. alone. Bacteria colonizing the catheters are highly adapted to the production of biofilms. Reasons as to why bacterial colonization of long term-inserted urethral catheters results in CA-UTI versus asymptomatic bacteriuria (CA-ASB) are essentially not known, and causative factors pertaining to the host environment and complexity of microbial biofilms may be implicated. Recent 16S rRNA gene surveys have indicated that microbial complexity of CA biofilms and urinary precipitates is higher than previously thought. The overall objective of this proposal is to characterize the dynamic formation and dispersal of these biofilms, as well as the triggers controlling relative human host inertia versus inflammatory responses. We hypothesize thata dynamic balance is established among pathogenic and lesser-characterized generally harmless bacteria, influencing the extent of the human host's immune response. A systems biology approach allows integration of diverse molecular datasets to elucidate signaling among microbial species responsible for cooperative as well as competitive behaviors and with the urothelial host defense system. The first Specific Aim is to profile the metagenome, metaproteome and metabolome of CA biofilms and dispersed bacterial aggregates from clinical cases in a longitudinal study design. The second Specific Aim is to develop and evaluate in vitro model systems inoculated with CA biofilm isolates and perform 'omics analysis on in vitro developing biofilms. The in vitro model systems will be based on cultivation of CA biofilm isolates with controlled level of oxygenation and synthetic urine as growth media. The third Specific Aim is to integrate and analyze metagenomic, metaproteomic and metabolomic datasets using advanced bioinformatics and multivariate statistics methods. We intend to identify biosignatures at five different levels: species, bacterial and host proteins and metabolites that characterize patterns of microbial communication with each other in the time domain and allow assessments of host tolerance towards and host defense against polybacterial colonization. We predict that the results will not only have profound implications as regards biofilm dynamics, but will also reveal biosignatures relevant in the context of diseases not limited to one infectious agent other than CA-UTI. 2.